A quantitative understanding of mass transport is an important aspect of successful drug development and drug efficacy. At the cellular and multi-cellular level, diffusion is the primary mechanism for the transport of drugs to cells, avascular tissues, and tumors1. However, transport is tightly controlled by numerous biological barriers including the plasma membrane, receptors, transport proteins, channels, vesicular systems, cell adhesion molecules, gap junctions, and cellular efflux pumps2,3. A drug's ability to cross cell and tissue barriers in vivo is a key determinant of the absorption, distribution, metabolism, excretion, and toxicity (ADME-Tox), and, ultimately, the success of the drug4-6. From the route of administration to the site of action, a drug encounters numerous biological barriers and drug transporters before reaching its intended target. The largest class of transporters is the family of ATP binding cassette (ABC) transporters. Much is known about one of these members, namely, p-glycoprotein (Pgp or ABCB1).
P-glycoprotein is a transporter that is localized to the plasma membrane of cells and is present in both normal and diseased tissues7,8. In normal tissues, Pgp helps to protect sensitive tissues from toxicity by facilitating efflux and preventing the intracellular accumulation of Pgp substrates8,9. For example, Pgp is constitutively expressed in the blood-brain barrier (BBB), the blood-testis barrier (BTB), and the placental barrier10-13. While the normal function of Pgp is protection from toxicity, it is also a significant barrier to drug transport and delivery. Pgp is constitutively expressed in the intestine, liver, and kidney, which decreases the bioavailability and distribution of drugs by hindering absorption through the intestine and increasing clearance into bile and urine5,8,14. Additionally, Pgp is up-regulated in diseased tissues and the cells of solid tumors. There, Pgp increases resistance to anti-cancer chemotherapeutics2,15. Unfortunately, many drugs of various pharmacological classes are substrates of this pump16.
Numerous inhibitors of Pgp have been identified, characterized extensively in vitro, tested in pre-clinical models, and evaluated in the clinic Although effective in vitro, Pgp inhibitors have been ineffective in the clinic, or have unexpected drug-drug interactions leading to increased toxicity7,18. There are three principal in vitro methods used to characterize inhibitors of Pgp: measurement of the efflux of radio-labeled compounds by a mono-layer of cells on a transwell dish, measurement of drug-stimulated ATPase activity of Pgp protein, and measurement of calcein-AM uptake by a mono-layer of cells19-22. Mono-layers of cells can measure inhibition of Pgp, but drug transport in these two-dimensional (2D) systems does not accurately replicate the complexity of the barriers found in a three-dimensional (3D) multi-cell layer environment. For example, the diffusion distance for a drug into a mono-layer of cells is relatively short compared to in vivo tissues and biological barriers are not adequately replicated in a 2D mono-layer. Moreover, there may be differential expression of Pgp in 2D culture versus 3D culture. Current methods to quantify 3D transport are cumbersome and include the use of microelectrode sensors to measure the concentration gradient of ions or monitoring the transport of radio-labeled molecules23,24. While these methods provide concentration profiles of a single plane through a tissue, they are time consuming and not amenable to higher throughput analyses because they rely on histological processing and contact autoradiography25,26.
Current in vitro models used to test the effects of inhibitors of Pgp often use single cells or mono-layers of cells such as MDR1-MDCK cells, a polarized kidney epithelial cell transfected to overexpress Pgp22. Using single cells and mono-layers, studies have quantified transport and reaction parameters such as diffusivity32, enzyme kinetics33, and drug inhibition34. Transport properties, molecular gradients, and cellular gradients have also been obtained from 3D models, but these require more complex experimental and analytical procedures such as two-photon microscopy35, incorporated probes36, tissue sectioning26, or mathematical models37,38. The 2D studies have also shown that verapamil (IC50=60.9±8.91), loperamide, cyclosporin A (IC50=2.2±0.02), and others are all effective inhibitors of Pgp at concentrations ranging from 1-100 μM using a calcein-AM assay, with percent maximum inhibition of 56.4%, 76.3%, and 98.7% respectivelyb 19,20.
Since drug efflux transporters are expressed at numerous locations within various organs and can transport a wide range of structurally diverse drugs, unwanted and unexpected side effects may occur when two or more therapeutic drugs are administered18. For example, despite being a potent opiate, loperamide administered alone does not cause opiate-like effects. However, co-administration of loperamide and quinidine (another Pgp inhibitor) increases the transport of loperamide across the blood brain barrier and leads to respiratory depression30. Conversely, drug efflux transporters are often up-regulated by solid tumors and contribute to resistance to chemotherapeutic agents2,15. In this case, more effective inhibitors of efflux transporters are needed to increase the concentration of the chemotherapeutic drug in the tumor. For example, Pgp drug resistance to several chemotherapeutic classes9,16, including vinca alkaloids, anthracyclines, and taxanes, has been observed in lymphoma, breast cancer, ovarian cancer, and small-cell lung cancer, as well as in tumors derived from tissues that constitutively express Pgp, such as colorectal cancer and renal cell carcinoma17,31. However, despite an important medical need, none of the well-known inhibitors of drug efflux transporters are currently used as an adjunct in the treatment of solid tumors either because they are clinically ineffective or they caused side effects, including increased toxicity, unwanted drug-drug interactions, or negative effects on the pharmacokinetics of the therapeutic15. Thus, there is a need for new in vitro models that can be used to predict potential toxicities and unwanted drug-drug interactions or to discover new and more effective inhibitors of drug efflux transporters.
Therefore, a need exists for a method and system to overcome or minimize the above-mentioned problems with the effect of candidate anticancer drugs on efflux pumps and gap channel communication.